It is known that 8-chloro-adenosine (8-Cl-Ado) is a novel RNA-directed nucleoside analog that targets leukemic stem cells (LSCs). In a phase I clinical trial with 8-Cl-Ado in patients with refractory or relapsed (R/R) AML, we observed encouraging but short-lived clinical responses, likely due to intrinsic mechanisms of LSC resistance. LSC homeostasis depends on amino acid-driven and/or fatty acid oxidation (FAO)-driven oxidative phosphorylation (OXPHOS) for survival. We recently reported that 8-Cl-Ado and the BCL-2-selective inhibitor venetoclax (VEN) synergistically inhibit FAO and OXPHOS in LSCs, thereby suppressing acute myeloid leukemia (AML) growth in vitro and in vivo. Herein, we report that 8-Cl-Ado inhibits ribosomal RNA (rRNA) synthesis through the downregulation of transcription initiation factor TIF-IA that is associated with increasing levels of p53. Paradoxically, 8-Cl-Ado-induced p53 increased FAO and OXPHOS, thereby self-limiting the activity of 8-Cl-Ado on LSCs. Since VEN inhibits amino acid-driven OXPHOS, the addition of VEN significantly enhanced the activity of 8-Cl-Ado by counteracting the self-limiting effect of p53 on FAO and OXPHOS. Overall, our results indicate that VEN and 8-Cl-Ado can cooperate in targeting rRNA synthesis and OXPHOS and in decreasing the survival of the LSC-enriched cell population, suggesting the VEN/8-Cl-Ado regimen as a promising therapeutic approach for patients with R/R AML.
Venetoclax (VEN) in combination with hypomethylating agents induces disease remission in patients with de novo AML, however, most patients eventually relapse. AML relapse is attributed to the persistence of drug-resistant leukemia stem cells (LSCs). LSCs need to maintain low intracellular levels of reactive oxygen species (ROS). Arsenic trioxide (ATO) induces apoptosis via upregulation of ROS-induced stress to DNA-repair mechanisms. Elevated ROS levels can trigger the Nrf2 antioxidant pathway to counteract the effects of high ROS levels. We hypothesized that ATO and VEN synergize in targeting LSCs through ROS induction by ATO and the known inhibitory effect of VEN on the Nrf2 antioxidant pathway. Using cell fractionation, immunoprecipitation, RNA-knockdown, and fluorescence assays we found that ATO activated nuclear translocation of Nrf2 and increased transcription of antioxidant enzymes, thereby attenuating the induction of ROS by ATO. VEN disrupted ATO-induced Nrf2 translocation and augmented ATO-induced ROS, thus enhancing apoptosis in LSCs. Using metabolic assays and electron microscopy, we found that the ATO+VEN combination decreased mitochondrial membrane potential, mitochondria size, fatty acid oxidation and oxidative phosphorylation, all of which enhanced apoptosis of LSCs derived from both VEN-sensitive and VEN-resistant AML primary cells. Our results indicate that ATO and VEN cooperate in inducing apoptosis of LSCs through potentiation of ROS induction, suggesting ATO+VEN is a promising regimen for treatment of VEN-sensitive and -resistant AML.
Note: BP, MV and LG, KG contributed equally Background Relapsed acute myeloid leukemia (AML) remains the most common reason for allogeneic hematopoietic cell transplant (HCT) failure. Thus, understanding AML immune escape mechanism is important for improving the odds of curing HCT patients with AML. Downregulation of HLA Class I and II expression by AML is one of the potential immune escape mechanisms. Therefore, treatment to restore HLA surface expression is crucial to prevent and treat relapse. Endogenous cytokines, such as IFN-γ, have been shown to stimulate HLA expression but are poorly tolerated by patients. However, two hypomethylating agents (HMA), decitabine (Dec) and azacitadine (Aza), that are routinely used in AML treatment are known to augment HLA expression. For AML, HMAs are often combined with venetoclax (Ven), a drug that blocks the anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein. Thus, while HMAs have been reported to increase HLA expression, what is unknown is whether these agents impact individual HLA loci differently and whether Ven has any impact on HLA expression. To address these questions, we treated the THP-1 cell line with Dec, Aza or Ven and measured changes in cell-surface expression of HLA proteins by flow cytometry using locus-specific HLA mAbs. Methods THP-1 cells were incubated with IFN-γ (500 U/mL), Aza (2µM), Dec (5µM), or Ven (30nM) for 48 hours (drug concentrations were determined by earlier titration experiments). THP-1 cells are a monocytic cell line, derived from the peripheral blood of a childhood case of acute monocytic leukemia (M5 subtype), that express HLA Class I and HLA-DR but not HLA-DQ or -DP under basal conditions, although they are inducible by IFN-γ. Thus, the induction of HLA Class II expression by IFN-γ serves as a positive control. Isotype controls were included to measure background. Data is presented as the difference in MFI (delta MFI) between cells treated with a drug and those treated with diluent only. Results Treatment of THP-1 cells with either IFN-γ or Dec led to increases in Class I HLA-A, -B & -C (Figure 1) compared to untreated cells (a mean fold increase of 1.4 and 1.2, respectively). Notably, Aza did not stimulate additional HLA-C expression and induced less of an increase in HLA-A & -B expression (an increase of 1.1-fold) than IFN-γ or Dec. Treatment of THP-1 cells by Ven did not induce a change in HLA Class I expression. For Class II, IFN-γ or Dec increased HLA-DR, -DQ and -DP expression in comparison to untreated cells (Figure 1). IFN-γ induced greater HLA-DR expression compared to Dec (an increase of 2.3-fold and 1.5-fold, respectively), and both stimulated similar increases in HLA-DQ (increases of 1.5-fold and 1.4-fold, respectively) & -DP (increases of 1.9-fold and 1.5-fold, respectively). However, treatment of cells with either Aza or Ven did not lead to changes in HLA Class II expression. Discussion Previous studies have illustrated the ability of IFN-γ to induce HLA Class II expression in THP-1 cells, however, data for Dec to induce HLA Class II expression was unconfirmed. We report differences in the degree to which IFN-γ and Dec are capable of stimulating HLA-DR with IFN-γ being more potent. The inability of Aza to induce HLA Class II expression in THP-1 cells may be related to the differing drug activating pathways of the two HMAs. Indeed, there are conflicting reports as to whether Aza can stimulate HLA Class II expression. Though Ven treatment of THP-1 cells did not impact HLA expression, because it is given with HMAs, it remains to be seen what effect these drugs may have on HLA expression when administered together. Additional studies to confirm these observations in patient-derived AML blasts are ongoing. Conclusion We report that HMAs increased expression of HLA-A, -B, & -C loci and Dec but not Aza stimulated HLA-DR, -DQ, and -DP expression in THP-1 cells. Given these data, Dec may be superior in increasing HLA Class II expression post-HCT. Figure 1 Figure 1. Disclosures Marcucci: Abbvie: Other: Speaker and advisory scientific board meetings; Agios: Other: Speaker and advisory scientific board meetings; Novartis: Other: Speaker and advisory scientific board meetings. Al Malki: Neximmune: Consultancy; CareDx: Consultancy; Jazz Pharmaceuticals, Inc.: Consultancy; Rigel Pharma: Consultancy; Hansa Biopharma: Consultancy.
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